0588-B3

Effect of seedborne Fusarium on nursery diseases of Pinus ponderosa Dougl. ex Laws in Argentina.

María Isabel Salerno ¹ Gladys Lori ² and Paula Morelli ³

Abstract

Seedborne Fusarium species have been reported on Pinus ponderosa Dougl. ex Laws in Western Patagonia, Argentina. The aim of our study was to determine the possibility of seed transmission of Fusarium species, relating their presence to damping-off and root diseases of P. ponderosa in forest nurseries. Plots containing a mixture of perlite, vermiculite and soil, were sterilized to eliminate residual inoculum and seeds were seeded without chemical seed treatments. Ten seedlings were grown per pot and replicated ten times. The incidence of damping-off was determined within two months. Seven Fusarium species were isolated from damped-off seedlings: F. equiseti, F. oxysporum, F. verticillioides (=F. moniliforme), F. incarnatum (=F. pallidoroseum=F. semitectum), F. sambucinum, F. acuminatum and F. proliferatum. These Fusaria were also detected on P. ponderosa seed lots. This is the first report on seedborne Fusarium species causing damping-off of P. ponderosa seedlings in Argentina. This shows that Fusariun species may initiate infections from seedborne inoculum demonstrating that seed transmission of Fusarium species is an important means by which soilborne diseases attacking P. ponderosa are spread within forest nurseries.

Introduction

Pinus ponderosa Dougl. Ex Laws. is one of the leading exotic tree species planted in Western Patagonia, Argentina (SubAntarctic region, next to the Cordillera de los Andes). Its potential for culture has long been recognized in the country and it has become economically important for both fiber and solid wood products' industries. This tree species is grown on 15- to 25-year rotations with yields higher than 30 m ³ /ha/ year (Mangieri et al. 1977).

A number of forest product companies have committed themselves to intensive, long-term programs of pine plantation management including tree improvement and nursery production. Numerous diseases attack conifer species in forest nurseries. Damping-off is the most common disease that affects seeds, germinants and young seedlings. The disease is caused mainly by soilborne pathogenic fungi among which Fusarium species have been considered particularly damaging (Bloomberg 1981, Lori et al. 2000). Seedborne fungi are considered the primary source of inoculum for damping-off and root rot fungi. The effects of seedborne fungi on the quality and quantity of germination of seeds of conifers have been the subjects of much debate and while some fungi are harmless, others cause considerable losses.

In Western Patagonia, several nurseries have experienced problems with damping-off and root rot caused by Fusarium spp. (Lori et al. 1999); some of these disease problems can result from infected seed. In order to gain insight into Fusarium species damaging conifer seed and affecting nursery production, we have studied the effects of Fusarium species on damping-off and root diseases and related their occurrence with Fusarium population associated with P. ponderosa seeds.

Materials and methods

Pinus ponderosa seedlots:

Seed samples from this test were harvested in eight different orchards located within the neighbouring forest nurseries in Western Patagonia. Details are presented in Table 1. Seeds samples were stored at 2-3°C during 40-60 days before each treatment. Seeds were placed in double plastic bags and they were then put back into stratification at 2-3°C until processed.

Fusarium on roots of diseased plants and on older seedling showing chlorosis symptoms

A biological assay which involved the cultivation of ponderosa pine seedlings for two months, on pots containing disinfected substrate composed of perlite, vermiculite and field nursery soil (2:1:1, v/v) was performed in the greenhouse under uncontrolled conditions. Post-emergence damping-off was determined by examining pots for fallen seedlings and disease incidence was expressed as the percentage of damped-off seedlings. Damped-off seedlings were carefully removed and washed in sterilized distilled water; the diseased roots were then surface sterilized by rinsing in 30% H₂ O₂ five seconds and plated on Potato Dextrose Agar medium (PDA) in order to determine the causal agents.

Following the experiment described above, three-month old seedlings were lifted and examined using a stereoscopic microscope to obtain their overall health. Twenty seedlings, emerging from each seedlot and showing foliar chlorosis symptoms, were harvested from their containers, and their roots analyzed for the presence of Fusarium species as described above.

Taxonomic identification of Fusarium species:

For the identification, Fusarium colonies were transferred to potato-dextrose agar medium (PDA); single spores were then isolated from each colony, plated on PDA medium and then incubated for 7-14 days at 22°C under fluorescent lamps supplemented with UV light during 12 hrs photoperiod. The micromorphology and culture features of the isolates were examined and identified according to the system of Booth and Nelson. In order to identify the Fusarium species within the section Liseola, the colonies were transferred to a ClK agar medium as described previously (Lori and Salerno, 2002).

Fusarium population associated with Pinus ponderosa seedlots:

Seed samples were assayed in order to detect Fusarium-infested seedlots. Ten seeds per plate were plated in a modified Nash and Snyder medium (Lori and Wolcan, 1996). Before plated, they were subjected to a running water rinse treatment for 24 or 48 h in order to speed germination and allow growth of fungi present in the seedcoats. The dishes were incubated at 22-25°C in the dark and monitored for the presence of fungi.

Results

Effect of Fusarium species on damping-off of P. ponderosa seedlings.

Damping-off was highest in seedlings emerged from seed collected from Mallín Ahogado , stored for one year (19%) and from Epuyén which was collected just prior to the assays (17%). The proportion of damped-off seedlings that came out from seed collected from Las Golondrinas was 15% whereas it was 13% on seed collected from Abra Ancha and Junín. The percentage of seedlings emerged from seed collected from Cerro Radal and El Maitén was lower (10%) compared to Cuesta del Ternero (12%).

Fusarium species associated with P. Ponderosa damped-off seedlings

A wide range of Fusarium species were isolated from damped-off seedlings emerging from P. ponderosa seedlots. F. oxysporum (10%) was the most abundant species isolated followed by F. equiseti (6%) and F. verticillioides (6%). F. incarnatum (=F. pallidoroseum=F. semitectum) (3%) and F. sambucinum (3%) were recovered in smaller quantities. F. acuminatum (2%) was only isolated from damped-off seedlings emerged from lot Las Golondrinas. Lot Las Golondrinas produce higher Fusarium counts (7%) compared to the other seedlots (Table 4)..

Isolations from root samples mainly yielded F. oxysporum (7%) and F. equiseti (5%). F. verticillioides (2%) and F. incarnatum (2%) were isolated in a lower frequency (Table 4).

Fusarium species recovered from Pinus ponderosa seed

Seven Fusarium taxa were recovered from the different lots of Pinus ponderosa seed: F. acuminatum, F. equiseti, F. oxysporum, F. incarnatum, F. verticillioides, F. sambucinum and F. proliferatum.

F. oxysporum (33%) was the most abundant species isolated from seed samples, followed by F. acuminatum (17%) , F. verticillioides (17%), F. incarnatum (13%), F. equiseti (11%). F. sambucinum and F. proliferatum were isolated in fewer quantities (5% and 3%, respectively). Fusarium counts were not completely eliminated following the water rinse treatments either for 24 or 48 h. In some cases, more Fusarium species were isolated after water rinses (Table 3).

Proportion of germinated seed of Pinus ponderosa

Germination results obtained on filter paper did not correspond closely to those obtained in the greenhouse. When seeds were plated on filter paper using the blotter method, germination was higher in lot Junin (44%) followed by lot Las Golondrinas (30%) and lots Epuyen (22.5%) and Abra Ancha (20%). Germination was very low in lots Cuesta del Ternero (11%), El Maiten (10%), Mallin Ahogado (7%) and Cerro Radal (3%). The proportion of germinated seed in the greenhouse was highest in Mallin Ahogado (76%) and Abra Ancha (65%) (Table 2).

Discussion

Our results show that many different Fusarium species are carried on seeds of P. ponderosa in Argentina that may serve as inoculum sources for damping-off and root rot diseases in forest nurseries. All Fusarium species isolated are common seedborne pathogens reported on conifer seeds around the world (Fraedrich and Miller 1995; Huang and Kuhlman 1990; Oak et al. 1999; Lori and Salerno, 2002). Fusarium population present on seeds had not been previously related to nursery diseases in Argentina. This is the first report of seedborne Fusarium associated with damping-off of P. ponderosa.

Seed with extensive Fusarium contamination was found in most lots. Running water rinses were not completely effective in reducing contamination by Fusarium, probably due to accelerated seed germination and proliferation of internal seed-borne fungi (Lori and Salerno 2002). Some Fusarium propagules were eliminated, or at least reduced which may have been carried superficially on the seed (Storer et al. 1998).

Seedcoat contamination with Fusarium spp. affected the performance of most Ponderosa pine seedlots and caused ungerminated seeds. Similarly, Pandey et al. (1990) reported Fusarium spp. associated with ungerminated seed of Pinus roxburgii. The reduced germination is often attributed to pre-emergence damping-off as Fusarium-contaminated seed produce weak germinants that die before the radicle emerges. Pre-emergence damping-off is difficult to diagnose because the affected seeds are not visible and the losses are often attributed to "poor seed" quality. Fusarium present on seeds also killed seedlings after the radicle of the seed has emerged indicating that seed quality is also an important factor involved in post-emergence damping-off. Among Fusarium species collected on seeds, F. oxysporum was the main pathogen recovered from most damped-off seedlings. F. proliferatum was not found causing damping-off although the fungus is a main pathogen of P. ponderosa (James, 1999).

Seeding characteristics varied with physiographic regions, tree conditions and/or stand conditions. Seedlots should be monitored to predict the appearance of nursery diseases which are difficult to diagnose during the germination and emergence stages and considering that an adequate source of high-quality seeds may not always minimize the impact of seedborne diseases in forest nurseries as the pathogens carried internally on seeds can be transmitted to the plant and cause damping-off diseases. Growers must be especially alert to prevent disease problems that can develop from seedborne inoculum and may spread quickly during early seedling development where succulent germinants may be killed within a few days. Prompt identification of Fusarium will determine chemical treatments to be applied before sowing to prevent damping-off occurrence or, as when the disease becomes evident, to prevent secondary spread.

Conclusions

It is important to consider that no matter how good seeds look, samples should be monitored to ascertain germinability and get an acceptable number of seedlings. Fusariun species are the primary source of inoculum and they interfere with the quality of planted seeds and the ability of crops to become established and realize their full potential of yield and value. Certain precautions during seed collection and handling practices should be taken to reduce the possibility of introducing seedborne pathogens into the nursery as seeds contaminated with Fusarium species, prechilled and stored may build up Fusarium counts. Significant changes have occurred in the global seed industry which include the greater importance of seed-transmitted pathogens in quality assurance programs, increased demands for international phytosanitary certificates for seed exports, concerns about dissemination of pathogens, improved seed treatment technology and application of biotechnology to seed disease control practices.

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Table 1. Seedlots of Pinus ponderosa collected from different seed orchards in Argentina.

Table 2. Seed germination in (i) filter paper (untreated seed) and (ii) pots containing growing medium after 1 and 3 months after sowing , expressed in percentage (%).

Table 3. Fusarium species isolated from Ponderosa pine seeds collected from different places after rinsing them with water.

Table 4. Fusarium species isolated from Ponderosa pine diseased seedlings and seeds collected from different places.

¹ CISAUA-Lab. Protección Forestal and ² Centro de Investigaciones de Fitopatología (CIDEFI-CIC) and Facultad de Ciencias Agrarias y Forestales, UNLP, 60 y 119 CC31 (1900) La Plata, Buenos Aires, Argentina.